AAV-S: A versatile capsid variant for transduction of mouse and primate inner ear

Abstract

Gene therapy strategies using adeno-associated virus (AAV) vectors to treat hereditary deafnesses have shown remarkable efficacy in some mouse models of hearing loss. Even so, there are few AAV capsids that transduce both inner and outer hair cells-the cells that express most deafness genes-and fewer still shown to transduce hair cells efficiently in primates. AAV capsids with robust transduction of inner and outer hair cells in primate cochlea will be needed for most clinical trials. Here, we test a capsid that we previously isolated from a random capsid library, AAV-S, for transduction in mouse and non-human primate inner ear. In both mice and cynomolgus macaques, AAV-S mediates highly efficient reporter gene expression in a variety of cochlear cells, including inner and outer hair cells, fibrocytes, and supporting cells. In a mouse model of Usher syndrome type 3A, AAV-S encoding CLRN1 robustly and durably rescues hearing. Overall, our data indicate that AAV-S is a promising candidate for therapeutic gene delivery to the human inner ear.

Keywords: AAV; adeno-associated virus vector; cochlea; gene delivery; gene therapy; hair cells; hereditary deafness; inner ear; non-human primate.

Graphical abstract

Figure 1

The AAV-S peptide is unlikely to distort capsid protein structure (A) Insertion point of AAV-S peptide in variable region VIII (VR-VIII) of the AAV9 capsid protein, with PHP.B insertion shown for comparison. (B) Peptide loop of AAV-S and PHP.B inserts. The structure of AAV9 VP3 (PDB: 3UX1) was modified with AAV-S or PHP.B residues and modeled using SWISS-MODEL to assess likely conformations of variants. (C) AAV-S and PHP.B loops in the context of VR-VIII, with AAV9 inset, modeled by SWISS-MODEL. Insertion is not predicted to affect capsid protein structure outside of VR-VIII, including the nearby VR-IV.

Figure 2

AAV-S efficiently transduces multiple cell types in the neonatal mouse cochlea (A) Schematic of the cochlea, with described regions numbered (see Table 1). Colored lines indicate the location of optical sections in the panels below. (B–H, J, and J′) Representative confocal images of whole-mount cochleas. C57BL/6J mice were injected via the round window membrane with AAV-S-CBA-EGFP at P1 with 3 × 10¹⁰ VG, and the cochleas were dissected and mounted at P6 (n = 8). (B) Confocal images of the apical, middle, and basal regions of the organ of Corti. The upper panel shows EGFP expression (green); the middle panel shows anti-MYO7A labeling for inner and outer hair cells (magenta), and the lower panel is a merged image. (C and D) Cochlea immunostained with anti-NF-H (magenta). Nerve fibers of the apical, middle, and basal regions (C) and cell bodies of spiral ganglion neurons (D) were transduced with AAV-S-CBA-EGFP (green, labeled with white asterisks). (E) Transduced interdental cells and fibrocytes of the spiral limbus (green). (F) Lateral wall, including EGFP-positive fibrocytes of the spiral ligament (green). (G) Outer sulcus cells and Claudius cells. Phalloidin labels filamentous actin (magenta). (H) Inner sulcus cells. (I and I′) Transduction efficiency in IHCs (I) and OHCs (I′) in C57BL/6J mice injected AAV-S-CBA-EGFP at P1 with 3 × 10¹⁰ VG. Bars indicate mean ± SEM (n = 8). (J and J′) Transduction by AAV-S-CBA-EGFP of hair cells and supporting cells in the macula of saccule; middle panels show anti-MYO7A labeling of hair cells. (J) is a top view; (J′) is a side view. (K) Auditory brainstem response (ABR) and (K′) distortion product otoacoustic emission (DPOAE) in wild-type C57BL/6J mice injected with 3 × 10¹⁰ VG of AAV-S-CBA-EGFP at P1. ABR and a DPOAE tests were performed at P25 for vector-injected (green; n = 3) and non-injected (black dashed; n = 3) animals. Bars indicate mean ± SEM. Scale bars, (B)–(H) 20 μm; (J) and (J′) 40 μm.

Figure 3

AAV-S-optiClrn1 delivery robustly and durably rescues hearing and morphology of hair bundles and auditory nerve fibers in the TgAC1⁺/ClrnKO mouse model of Usher 3A (A and B) ABR and DPOAE thresholds at P35, P60, P90, P120, P150 of wild-type C57BL/6J mice (black dashed; n = 3–9) and TgAC1⁺/Clrn1KO mice, either untreated (green; n = 3–6) or treated at P1 with AAV-S-optiClrn1 (1.9 × 10¹⁰ VG) (magenta; n = 3–12). Untreated TgAC1⁺/Clrn1KO mice show some residual auditory sensitivity at P35 that is largely absent by P60. Treated mice retain near-wild-type sensitivity at low and middle frequencies to at least P150. Error bars indicate mean ± SEM. (C) Representative scanning electron micrographs of hair bundles of the cochlea. All images were collected from the mid-cochlear region at P60 or P150. (C and D) Wild-type C57BL/6J mice (n = 3–4). (C′ and D′) Untreated TgAC1⁺/Clrn1KO mice (n = 3–5). (C″ and D″) Mice treated at P1 with AAV-S-optiClrn1 (1.9 × 10¹⁰ VG) (n = 3–4). Missing hair bundles are labeled with white asterisks; surviving but severely disorganized hair bundles are labeled with black asterisks; black arrow points to a loss of short-row stereocilia; white arrow points to a loss of middle-row and short-row stereocilia. Scale bars, upper panels 5 μm; middle and lower panel, 1 μm. (E) Confocal images of OHC bundles at P90 labeled with phalloidin (yellow). Wild-type C57BL/6J mice (left panel, n = 3). Untreated TgAC1⁺/Clrn1KO mice (middle panel, n = 4). TgAC1+/Clrn1KO mice treated at P1 with AAV-S-optiClrn1 (right panel, n = 3). Scale bar, 5 μm. Bundles are disorganized in untreated mutant animals and largely normal with treatment. (F) Confocal images of the apical, middle, and basal regions of whole-mount cochleas at P90 immunostained with anti-MYO7A (magenta) to label hair cells and anti-NF-H (cyan) to label cochlear nerve fibers. Cochlear innervation is markedly reduced in untreated mutant animals (middle panel, n = 4) and retained with treatment (right panel, n = 3). Scale bar, 20 μm.

Figure 4

AAV-S mediates robust transgene expression in a variety of cell types in the NHP cochlea and saccule (A–D) Representative low-magnification images of frozen sections (18 μm thick) of the apical, middle, and basal regions of the cochlea. (A and B) Cochleas injected with 4.7 × 10¹¹ VG (n = 2 ears). (C) Cochlea injected with 8 × 10¹⁰ VG (n = 1 ear). (D) Control ear injected with PBS (n = 1 ear). In each, the right panel shows anti-EGFP labeling (green); the left panel also shows anti-MYO7A labeling (magenta) and DAPI labeling (blue) superimposed to visualize hair cells and nuclei. (E) Whole-mount images of the apical, middle, and basal regions of cochlea injected with 5.8 × 10¹¹ VG (n = 1 ear). (F) Summary schematic of AAV-S transduction (green) in the cochlea. Specific regions are numbered (see Table 1). (G and G′) Transduction efficiency in IHCs and OHCs from apex to base in an NHP cochlea injected with 5.8 × 10¹¹ VG (n = 1 ear) and 4.7 × 10¹¹ VG (n = 2 ears) of AAV-S-CBA-EGFP. Error bars indicate mean ± SEM (H and H′) Frozen sections (18 μm thick) of the utricular macula (H) and saccular macula (H′) in an inner ear injected through the RWM with 4.7 × 10¹¹ VG (n = 2 ears). The upper panels show a superposition of EGFP and MYO7A labels, along with phalloidin (blue) to mark actin of the hair bundles. Scale bars, (A)–(D); 200 μm, (E) 30 μm; (H) and (H′) 50 μm.

Figure 5

Robust transgene expression is observed in the organ of Corti and vestibule in NHPs injected with AAV-S-CBA-EGFP (A) High-magnification images of frozen sections (18 μm thick) of the apical, middle, and basal regions of the organ of Corti in a cochlea injected with 4.7 × 10¹¹ VG (n = 2 ears). The left panel shows AAV-S-transduced cells, labeled with an antibody to GFP (green); the middle panel shows hair cells labeled with an anti-MYO7A antibody (magenta), and the right panel shows both, along with DAPI labeling of nuclei (blue). (B) Schematic of the organ of Corti. Green indicates EGFP expression mediated by AAV-S. Specific regions are numbered (see Table 1). (C–C″) High-magnification images of a whole mount of a cochlea injected with 5.8 × 10¹¹ VG (n = 1 ear). (C) AAV-S-CBA-EGFP transduces Hensen’s and Claudius cells (green). (C′) AAV-S-CBA-EGFP transduces IHCs, OHCs, Deiters’, and pillar cells. (C″) AAV-S-CBA-EGFP transduces inner sulcus epithelial cells. (D) The lateral wall of a cochlea injected with a high dose of AAV-S-CBA-EGFP in an 18-μm frozen section. (E) The top view and side view of the spiral ligament, showing labeled fibrocytes. (F and F′) High-magnification images of a frozen section of the utricular macula (F) and saccular macula (F′) in an inner ear injected with 4.7 × 10¹¹ VG. eGFP colocalizes with anti-MYO7A labeling of hair cells, indicating that most hair cells were transduced (white). Scale bars, (A), (D), and (E), 200 μm; (C)–(C″) and (F), 30 μm.

Figure 6

NHPs injected with AAV-S-CBA-EGFP show minimal immune infiltration in the inner ear (A) Hematoxylin and eosin-stained sections of a NHP cochlea injected with 4.7 × 10¹¹ VG of AAV-S-CBA-GFP. Minimal focal perivascular mononuclear cell infiltration was observed (black arrows). (B) Hematoxylin and eosin-stained sections of a NHP cochlea injected with PBS. Scale bars, 50 μm.

Figure 7

ABR testing of NHPs before and after injection of AAV-S-CBA-EGFP showed no loss of sensitivity for most injections NHPs were tested with click and tone ABR before AAV-S vector injection or PBS control injection and then three weeks later before euthanasia. (A–A″) Saline injection control (n = 1 ear). (B–B″) Low-dose AAV-S (n = 1 ear). (C–D″) High-dose AAV-S (n = 2 ears). Click ABR raw traces before (A–D) and after (A′–D′) injection. (A″–D″) ABR thresholds in response to pure tones of 500, 1,000, 2,000, and 4,000 Hz and to clicks, before (solid) and after (dashed) injection. ABRs are plotted on an inverted scale as for human audiology, so hearing loss (higher threshold) is shown as down.